Role of third intracellular loop of galanin receptor type 1 in signal transduction

Role of third intracellular loop ofgalanin receptor type 1 in signaltransduction

K. Rezaei, 1 K. Saar, 1,2 U. Soomets, 1,2 A. Valkna, 1,2 J. Näsman, 3 M. Zorko, 1,4

K. Åkerman, 4 T. Schroeder, 1 T. Bartfai, 1,5 Ü. Langel 1

1Department of Neurochemistry and Neurotoxicology, Stockholm University, S-10691 Stockholm, Sweden, 2Department of Biochemistry, University of Tartu,51014, Tartu, Estonia, 3Department of Physiology, University of Uppsala, S-75123 Uppsala, Sweden, 4Institute of Biochemistry, Medical Faculty, University ofLjubljana, 1000 Ljubljana, Slovenia, 5F. Hoffmann-La Roche Ltd, Department PRPN, CH-4070 Basel, Switzerland

Summary To determine the domains essential for G-protein coupling of the human galanin receptor type 1 (GalR1),we have used both GalR1 mutants and synthetic receptor-derived peptides in 125I-galanin and [35S]-GTPγS bindingstudies. Replacement of potential phosphorylation sites by Leu in the third intracellular loop (IC3) of GalR1 did notaffect KD values for the receptor. Peptides derived form the IC3 loop, and especially the N-terminal part of it were ableto increase the rate of [35S]-GTPγS binding to the trimeric Giα1β1γ2, but not to Gsαβ1γ2, whereas the peptidescorresponding to the IC1 and IC2 loops had no such effect. IC3 loop peptides also inhibited the binding of 125I-galaninto GalR1 in membranes from Rin m5F cells. Our results suggest that the IC3 loop of GalR1, especially its N-terminalpart, defines the coupling of the receptor to the Giα1β1γ2 protein and consequently, to the signal transduction cascade.© 2000 Harcourt Publishers Ltd

Neuropeptides (2000) 34 (1), 25-31© 2000 Harcourt Publishers Ltd

DOI: 10.1054/npep.1999.0782, available online at http://www.idealibrary.com on

INTRODUCTION

Galanin, a neuropeptide of 29 amino acids (30 inhuman), was originally isolated from upper porcine intes-tine at Mutt’s laboratory in 1983.1 It is widely distributedin both the central and peripheral nervous systems andmodulates several important physiological functions.2

Galanin is involved in the regulation of learning andmemory3, feeding4, sexual behaviour5, release of insulin,acetylcholine, dopamine, growth hormone, and prolactin,as well as mobility of digestive tract, and pain signalling6,and furthermore is suggested to be involved in thepathogenesis of Alzheimer’s disease.7 The wide range ofphysiological roles of galanin in different tissues makesgalaninergic signal transduction interesting from a thera-peutical point of view. There is evidence that galaninmediates various physiological effects through distinctreceptor subtypes.2

Received 12 July 1999Accepted 12 November 1999

Correspondence to: Ülo Langel, Department of Neurochemistry andNeurotoxicology, Stockholm University, S-10691 Stockholm, Sweden.Tel.: 46 8 161 793; Fax: 46 8 161 371; E-mail: [email protected]

The first galanin receptor subtype (GalR1) was clonedfrom human melanoma Bowes cell line in 1994.8 In fol-lowing years GalR1 was cloned from human brain9,human small intestine10, rat brain11, and the rat insuli-noma cell line Rin 14B.12 The second galanin receptorsubtype, GalR2 was first cloned in 1997 followed bycloning of a third subtype, GalR3. The GalR2 amino acidsequence has 40% homology with GalR1 and 54% withGalR3, while GalR1 and GalR3 share 36% homology.13–16

The low degree of similarity between the GalR subtypesas compared to the other receptor families may dependon the relatively large galanin molecule that containsseveral distinct epitopes for recognition by differentreceptor subtypes.

The first evidence of interaction between GalRs andpertussis toxin sensitive G-proteins was shown by ligandbinding studies in rat hippocampal tissue and Rin m5Finsulinoma cells.6,17 All three receptor subtypes are themembers of the superfamily of G-protein-coupled recep-tors which transduce their signals by activating a varietyof intracellular second messenger systems, includinginhibition or stimulation of cAMP formation, inhibitionof phospholipase C activity, blockage of voltage-depen-dent Ca2+ channels, and activation of ATP-dependent K+

25

26 Rezaei et al.

channels.6 Activation of the GalR1 inhibits basal andforskolin-stimulated cAMP formation in several celltypes, while the activation of the GalR2 stimulates phos-phatidylinositol turnover and mobilization of intracellu-lar Ca2+.

The structure of both human and mouse GalR1 geneshas been studied.18,19 Three exons were identified in theopen reading frame encoding both the human andmouse GalR1. The second exon encodes the third intra-cellular loop of the receptor, a region that has beenshown to have a major role in coupling of several hepta-helical receptors to heterotrimeric G-proteins such asadrenergic, muscarinic, and dopaminergic receptors.20–23

We have used mutagenesis studies and synthetic pep-tides to determine functionally important amino aciddomains in the intracellular loops of several transmem-brane receptors for G-protein interaction.24–26

Studies with synthetic peptides that mimic the cyto-plasmic structures of receptors suggest that these loopsdirectly interact with G-proteins. For example, the pep-tides mimicking the second and third cytoplasmic loopsof rhodopsin and part of its carboxyl-terminus bind tothe rod cell G-protein, transducin, and block the receptorcoupling.27 All these reports have identified amino acidsand sequences in the N- and C-terminal parts of the IC3which are involved in G-protein interactions.

Activation of the GalR1 results in the inhibition of per-tussis toxin-sensitive forskolin-stimulated cAMP forma-tion in several cell types, indicating coupling of thisreceptor to heterotrimeric Gi/o proteins. The coupling toGi/o may be mediated partly by IC3 of the receptor, sinceIC3 corresponding peptides potently inhibit basal adeny-late cyclase and GTPase activity in rat ventral hippocam-pal membranes in the same manner as a galanin receptoragonist.28

The aim of the present study was to identify cytoplas-mic domains responsible for the human GalR1 G-proteininteraction. Receptor mutants and synthetic peptides cor-responding to the whole and parts of the cytoplasmicloops of human GalR1 were constructed, and their effecton 125I-galanin binding as well as [35S]-GTPγS binding tothe membranes from different cells were studied.

MATERIALS AND METHODS

Peptide synthesis

The peptides were assembled on the model 431A peptidesynthesiser (Perkin Elmer Applied Biosystems Inc., FosterCity, CA, USA) using tert-butyloxycarbonyl (t-Boc) strat-egy of solid phase peptide synthesis. To obtain C-termi-nally amidated peptides, tert-4-methyl-benzhydrylamineresin was used. Dicyclohexylcarbodiimide/N-hydro-xybenzotriazole activation strategy was applied for

Neuropeptides (2000) 34(1), 25–31

coupling of all protected amino acids except t-Boc-gluta-mate (OcHex) which was coupled using the 2-(1H-benzo-triazole-1-y1) 1,1,3,3-tetramethyluronium tetrafluroborate/N-hydroxybenzo-triazole activation method. Aminoacids, activators and resin were from Bachem AG(Bubendorf, Switzerland). Deprotection of the side-chains, cleavage of the peptides from the resin and purifi-cation followed the protocol described by Langel et al.29

Molecular mass of each synthetic peptide was deter-mined using Plasma Desorption Mass Spectrometer(Bioion 20, Uppsala, Sweden) and the calculated valueswere obtained in each case.

Expression of G-proteins in Sf9 cells

Baculovirus transfer vectors harbouring the genes forGsα, Giα1 and β1γ2 were kindly provided by Dr TatsuyaHaga (University of Tokyo, Tokyo, Japan). The cDNA forbovine Gsα30 in pVL1392, bovine Giα131 in pVL1392 andbovine β1γ232 in pVL1393 were cotransfected with lin-earized baculovirus DNA (Pharmingen, San Diego, CA,USA), and the resulted virus stocks were subjected to oneround of plaque purification before generation of high-titer virus stocks. In the expression procedure, Sf9 cells ata density of 2 million cells/ml in suspension culture wereinfected with a ratio 2:1 of α versus βγ. Cells were har-vested 48 h post-infection, washed in phosphate-bufferedsaline and stored at –70°C until membrane preparation.

Membrane preparation for [ 35S]-GTPγS binding assay

Frozen Sf9 cells overexpressing Giα1β1γ2 or Gsαβ1γ2were thawed and resuspended in 5 volumes of ice-coldTE-buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 7.5).Obtained suspension was homogenised and then passedthrough a syringe needle (26-G). Homogenate was cen-trifuged at 500 × g for 15 min, the pellet discarded andthe supernatant centrifuged at 40 000 × g for 40 min.Obtained pellet was washed in 10 volumes of TE bufferand re-centrifuged at 40 000 × g for 40 min. Resultingpellet was resuspended in TE buffer to give a protein con-centration 2–4 mg/ml. Aliquots of the obtained mem-brane preparation were kept at –70°C until the day ofexperiment.

[35S]-GTPγS binding assay

The effect of the peptides on the initial rate of [35S]-GTPγSbinding was determined by following the protocol pre-sented by McKenzie33 with minor modifications. Briefly,the membranes (the final protein concentration in theassay mixture was 0.5 mg/ml) were incubated with 0.5 nM[35S]-GTPγS (Du Pont-NEN, Boston, MA, USA) at30°C in assay buffer (10 mM Tris-HCl, 0.1 mM EDTA,

© 2000 Harcourt Publishers Ltd

Galanin receptor type 1 in signal transduction 27

Fig. 1 Schematic diagram of human galanin receptor, GalR1,based on the cDNA sequence, showing the locations of aminoacids mutated in this study (in black).

5 mM MgCl2, 150 mM NaCl, 1 mM DTT, 1 µM GDP, pH7.5). The unbound [35S]-GTPγS was removed by rapid fil-tration of the reaction mixture through CF/G glass fiberfilters (Whatman International Ltd, Maidstone, UK).Radioactivity retained on the filters were determined in aPackard 3255 liquid scintillation counter (PackardInstrument Company, Meriden, CT, USA).

Point mutations in the third intracellular loop

All single amino acid mutations were introduced by thepolymerase chain reaction (PCR) overlap technique usingtwo sets of primers (Medprobe A/S, Oslo, Norway) onpRK8 plasmid34 carrying GalR1 cDNA as a template,using Pfu polymerase (Stratagene, Texas, USA). In allcases a diagnostic cleavage site was introduced to facili-tate the screening of recombinants. After the final roundof PCR with external primers, an extended PCR productcarrying the mutation was double digested with BstX1and Not1 (New England Biolabs, Boston, USA) restricationenzymes. The cleavage product corresponding to thefragment of the expected size was excised and elutedfrom agarose gel using a Jetsorb kit (Genomed, Hannover,Germany). After ligation of PCR products into the vectorwith T4 ligase (New England Biolabs) and transformationof E coli strain XL1-Blue, recombinants were selected byrestriction analysis. Plasmid corresponding to the correctmutant was purified by an alkali lysis method using aQiagen kit (Qiagen GmbH, Hilden, Germany). All muta-tions were verified by DNA sequencing around theexpected positions of mutations.

Transient expression of human GalR1 cDNA constructs

For the transient expression of wild-type and humangalanin receptor mutants, COS-7 cells were grown to50–70% confluency in Dulbecco’s Minimal Essentialmedium (GIBCO, Scotland, UK) containing 10% (v/v) fetalcalf serum, 2 mM L-glutamine, 100 U/ml penicillin, and100 mg/ml streptomycin in 5% CO2 (v/v) enriched air at37°C. For binding studies, 20 µg plasmid DNA per dishwere transfected by calcium phosphate precipitation. Cellswere used in binding studies 40–48 h after transfection.

Binding experiment using Rin m5F cell line

Forty-eight h after transfection, the transfected cells wereharvested with a rubber ‘policeman’ and pelleted at 1000 × g for 10 min at 4°C. The cells were exposed tohypoosmotic shock in 5 mM Tris-HCl buffer (pH 7.4) onice for 30 min. The suspension was centrifuged at 10 000× g for 45 min at 4°C and the resulting pellet was resus-pended in 5 mM Hepes buffered Krebs-Ringer solution,KRH (137 mM NaCl, 2.68 mM KCl, 2.05 mM MgCl2, 1.8


mM CaCl2, 1 g/l glucose), pH 7.4, supplemented with0.05% (w/v) bovine serum albumin (BSA), 0.02 mM 1,10-phenanthroline, and used immediately in equilib-rium binding experiments. The equilibrium bindingexperiments were performed in a final volume of 400 µlKRH, containing 0.02 mM 1,10-phenanthroline, 0.1–0.2 nM porcine 125I-galanin (DuPont-NEN), 80 µltransfected COS-7 cell membrane preparation and vary-ing concentrations of unlabelled galanin or loop peptide.Samples were incubated for 30 min at 37°C. The incuba-tion was terminated by the addition of 2 × 10 ml of ice-cold KRH, followed by rapid filtration over WhatmanGF/C filter precoated for 2–3 h in 0.3% (v/v) polyethylen-imine (Sigma) solution. Radioactivity retained on the filters was determined in a Packard γ counter.

RESULTS

Mutation of potential phosphorylation sites in IC3 loopof GalR1

There are three serine and one threonine residues withinthe IC3 loop of GalR1 which are potential sites for kinase-mediated phosphorylation. To determine the importanceof these residues, point mutations of GalR1 were per-formed. Three Ser and one Thr residues were indepen-dently substituted with a Leu residue (Fig. 1). COS-7 cellswere transfected with the pRK8 vector containing cDNAof S235L, S238L, S241L and T245L mutant receptors (Fig. 1). Two days after transfection, binding studies with

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28 Rezaei et al.

100

80

60

40

20

0

B/B0%

Ñ10 Ñ9 Ñ8 Ñ7 Ñ6 Ñ5log [galanin], (M)

Fig. 2 Displacement of porcine 0.2 nM 125I-galanin by humangalanin in membrane preparations of COS-7 cells transfected withfollowing plasmids encoding native hGalR1 and its mutants: pRK8-hGR (wild type, –m–), pRK8-hGR[S235L] (–²–)), pRK8-hGR[S238L] (–✚–)), pRK8-hGR[S241L] (–o–)) andpRK8-hGR[T245L] (–×–)).

Fig. 3 Schematic representation of the GalR1. The solid lineshows the synthetic peptide sequences synthesized and studied inthis work.

125I-galanin showed only a slight reduction in maximalbinding level for the mutants as compared to the wildtype receptor. The mutations did not influence the affin-ity of the receptor for galanin significantly, yielding in thedissociation constants (KD) ranging from 0.7 to 3 nM (Fig.2). This result indicates that these particular Ser and Thrresidues do not play a role in uncoupling of the GalR1receptor from galanin.

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Table 1 Effects of cytoplasmic loop-peptides on membrane preparations from Sf9 cells expressing

Peptide Efficac

IC1W(58–72) no effeLARSKPGKPRSTTNLFamideIC2W(132–153) no effeDRYVAIVHSRRSSSLRVSRNALamideIC3W(222–245) maximal actKVLNHLHKKLKNMSKKSEA 34 ± 3%SKKKTamideIC3N(222–233) maximal actKVLNHLHKKLKNamide 42 ± 4%IC3C(235–245) no effeSKKSEASKKKTamide

Influence of GalR1 mutated peptides on binding of125I-galanin to membranes from Rin m5F cells

Five peptides corresponding to the whole of GalR1 IC3loop were synthesized (Table 1). The following aminoacids; Ser at position 235, 238, 241 and Thr at position245, were replaced by Leu in one peptide each and a fifthpeptide was synthesized without any replacement. Incontrast to the whole IC3, these mutated peptides apart from IC3(S238L) did not affect the binding of 125I-galanin to membranes from Rin m 5F cell (Table 2).


the initial rate of [35S]GTPγS binding to Giα1β1γ2 and Gsαβ1γ2 respectively

Giα1β1γ2 Gsαβ1γ2

y EC50(nM) Efficacy

ct no effect no effect


ivation 0.17 ± 0.07 no effect

ivation 0.35 ± 0.19 not tested



Table 2 Inhibition of specific 0.2 nM 125I-galanin binding by cyto-plasmic loop-peptides and their analogs in Rin m5F cells

Peptide (100 µM) Ki(app) µM

ICI >100IC2 >100IC3 38 ± 26IC3(S238L) 75 ± 37IC3(S235L) >200IC3(S241L) >200IC3(T245L) >1000

The IC3(S238L) peptide showed 2-fold lower value ofKI(app) on binding as compared to the whole IC3 peptide.This result suggests that the Ser 235 and 241, Thr 245and, to a lower extent, the Ser 238 are important for thecompetition of the whole IC3 peptide with receptor toblock GalR1-G protein interaction.

Effect of GalR1 peptides on [ 35S]-GTPγS binding inGiα1β1γ2 and Gs αβ1γ2 on membranes from Sf9 cells,and influence of these peptides on 125I-galanin binding

We have synthesized the GalR1 derived peptides corre-sponding to the whole IC1, IC2 and IC3 loops, the N-ter-minal parts of IC2 and IC3, as well as the C-terminal partsof IC2 and IC3 (Fig. 3). The influence of these peptides on125I-galanin and [35S]-GTPγS binding on membranes fromRin m5F cells as well as on [35S]-GTPγS binding on mem-branes from Sf9 cells expressing Giα1β1γ2 and Gsαβ1γ2proteins were studied. The whole and N-IC3 loop pep-tides increased the rate of [35S]-GTPγS binding to themembranes from Sf9 cells transfected with Giα1β1γ2,while no activity was observed with the C-IC3 loop pep-tide (Table 1). The maximum rate of [35S]-GTPγS bindingas compared to the basal level was 34±3% and 42±4%respectively. In Rin m5F cell membranes, the maximalactivating effect of the whole IC3 peptide was 16±4% as compared to the basal activity. Syntheticpeptides corresponding to the whole IC3 of GalR1 werenot able to influence [35S]-GTPγS binding in membranesfrom Sf9 cells expressing Gsαβ1γ2 (data not shown).These data suggest that the whole of IC3 peptide andespecially the N-terminus of this loop participate inGalR1-G protein interaction.

Receptor derived peptides corresponding to the wholeIC1, whole IC2, N-IC2, and C-IC2 had no effect on 125I-galanin binding to the galanin receptors in Rin m5F cellmembranes, whereas the peptide derived from the wholeIC3 loop inhibited ligand binding with Ki(app) value of 38µM (Table 2). This is consistent with our previous resultsobtained on membranes from rat hypothalamus andBowes melanoma cells.28


DISCUSSION

In the present study, we utilized both synthetic peptidesand mutagenesis strategy to study a possible involve-ment of IC loops and especially the IC3 loop of the GalR1,in its coupling to G-proteins. We measured the effect ofthe peptides on 125I-galanin and [35S]-GTPγS binding indifferent cell lines. We clearly show that IC3 loop pep-tides affect both 125I-galanin and [35S]-GTPγs binding indi-cating that the IC3 loop is involved in Gα-proteinactivation. According to these data the IC3 peptide hadthe highest inhibitory effect on 125I-galanin binding withKi(app) value of 38 µM (Table 2), whereas IC1 and IC2 looppeptides did not show any significant effects. Our resultsare consistent with previously described effect of theGPCR synthetic peptides on agonist binding for, e.g. δ-opoid receptor35 and α2-adrenergic receptor.36 Thisinhibitory effect supports the idea that these intracellularpeptides mimic the receptor-G-protein interaction. Thedisruption of coupling between the Ga1R1 and the ago-nist by a specific synthetic peptide supports the notionthat a high affinity component of agonist binding occurswithin a ternary complex consisting of agonist, receptorand G-protein.37

The whole and N-IC3 loop peptides increased the rateof [35S]-GTPγS binding to membranes from Sf9 cells trans-fected with Giα1β1γ, while no activity was observed withthe C-IC3 loop peptide (Table 1). The maximum rate of[35S]-GTPγS binding in the presence of the whole and N-IC3 loop peptides as compared to the basal level was 34%and 42% respectively. This finding indicates that thesepeptides activate GalR1 by accelerating the GDP/GTPexchange. This was confirmed by the observation thatthe whole IC3 and N-IC3 peptides could stimulate [35S]-GTPγS binding to activate Giα1β1γ1 protein. The similarobservation was reported by Varrault and coworkers.38

It is commonly believed that agonist-induced receptordesensitization involves phosphorylation of the agonist-bound form of the receptor by G protein receptor kinases(GRKs) causing uncoupling of the activated receptorfrom its G protein.39–41 The third intracellular loop of var-ious G protein-coupled receptors contain several Ser andThr residues that are potential phosphorylation sites ofGRKs. For the δ-opoid receptor the Thr 353 in the IC3loop has been shown to be important for receptor desen-sitization and internalization.42,43 Also, in µ-opoid recep-tor and in platelet activating factor receptor, Ser and Thrresidues have been identified to be involved in receptordesensitization44–46. In our site-directed mutagenesisstudies, the replacement of Ser and Thr residues withinthe IC3 loop with Leu showed no increase in cell surfacereceptor binding by agonist as compared to the wild-typereceptor. On the other hand, in contrast to the whole IC3mutated peptides from IC3, those mutated peptides apart

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30 Rezaei et al.

from IC3(S238L) could not affect the binding interactionof 125I-galanin (Table 2). This may be due to the fact thatin the complete receptor the IC3 loop is anchored totransmembrane regions, which contribute to the struc-tural stability of the IC3 loop. This feature is lacking insynthetic peptides and may explain the apparent discrep-ancy in sensitivity of mutated receptors and peptides.

In conclusion, two peptides, one corresponding to thewhole IC3 loop and the other to the N-terminal part of itinhibited the binding of 125I-galanin to GalR1 in Rin m5Fcells suggesting that the IC3 loop of GalR1, especially itsN-terminal part defines its coupling to Giα1β1γ2 proteinsubunit and links it to signal transduction cascade.Substitution of the Ser and Thr residues in the IC3 loopof GalR1 which are potential phosphorylation sites donot seem to be important for the ability of IC3 to interactwith the G-protein.

ACKNOWLEDGEMENTS

This work was supported by grants from the SwedishResearch Council for Natural Sciences (NFR) and fromEngineering Sciences (TFR), BMH4 CT95 0172 of theEuropean Community.

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Neuropeptides (2000) 34(1), 25–31

Role of third intracellular loop of galanin receptor type 1 in signal transduction

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